DESCRIPTION: Porphyromonas gingivalis (P. g.), a Gram-negative anaerobe is known to play a critical role in the development of periodontitis. Several lines of evidence had also indicated its role in the development of cardiovascular disease (CVD). First, epidemiological studies have concluded that individuals with periodontitis were at greater risk for CVD. Second, 16S rDNA of P. g. was found to be present in atheromatous plaques. In vitro, P. g. has been shown to invade human coronary artery endothelial cells (HCAEC) and coronary artery smooth muscle cells (CASMC). In vivo, the long-term systemic challenge of Pg can accelerate atherogenic plaque progression in apolipoprotein E-deficient mice. Recently, we have found the presence of live bacteria in atherosclerotic tissue from patients. All these observations suggest that P. g. may invade cardiovascular tissues, thus contributing to the development of atherosclerosis by interfering with cellular function of host tissues and by eliciting an inflammatory response. Therefore, the central hypothesis is that P. g. contributes to the development of atherosclerosis by invasion of tunica intima and intima media. The Specific Aims of this study are to probe the ability and mechanism of cardiovascular tissue invasion and penetration by P.g. using novel human organ and cell culture systems. Human saphenous vein organ culture that maintains 3-D structure of vascular tissue will be used for the first time to evaluate invasion and penetration ability of P.g. The depth and amount of invaded P.g in tunica intima and intima media will be determined by real time quantitative PCR and immunofluorescent microscopy using antibodies against P.g., endothelial and smooth muscle cell markers. By this system, we will 1) determine whether invasion can lead to neoinitima formation. 2) compare the invasion ability in injured and non-injured veins. 3) determine the relationship of demographic and medical data of vein donors with the susceptibility of the invasion. To probe the transcellular or/and paracellular route(s) of tissue penetration by P.g., primary endothelial and smooth muscle cell lines will be co-cultured in a mono-layer or bi-layer transwell culture system in which endothelial cells will be kept in transwell inserts, resembling in vivo layout. The ability of P.g. to either exit the pre-infected endothelial cell layer or penetrate the un-infected endothelial cell layer to invade smooth muscle cells will be evaluated. In parallel, the endothelial cell layer morphology and integrity will be monitored by scanning electronic microscope and the status of endothelial cell junctions will be detected by measuring transendothelial resistance. The proposed experiments will determine whether P.g can invade and penetration vascular tissue through transcellular or/and paracellular pathway(s). The completion of this study will contribute to the long-term goal of understanding this bacterially induced vascular inflammation and identification of new therapeutic targets. [unreadable] [unreadable]